The dynamics of secretory vesicles in living hyphae of the pathogen Ustilago maydis.

丝状真菌是一类在进化上取得成功的具有巨大生态意义的生物，是菌根与植物相互作用的共生体，也是植物垃圾的分解者。它们用于蛋白质的工业生产，并作为病原体对公共卫生和农业构成威胁。丝状真菌的基本单位是菌丝；菌丝通常由一串细长的细胞组成，这些细胞在顶端通过扩张而生长，这一过程称为顶端生长。这种生长模式允许组织和底物的入侵。菌丝顶端的生长需要新合成的膜、蛋白质和细胞壁前体在一个称为分泌的过程中持续供应给菌丝顶端。这些补给的载体是分泌运输囊泡，被带到顶端，在那里它们与质膜融合。在这项赠款申请的背景下，重要的是要注意到，我们目前对分泌的看法是，高尔基体后小泡单向运动到菌丝顶端，在那里它们与质膜融合，从而传递膜和蛋白质。分泌小泡被运送到菌丝顶端的机制尚不清楚。类似于其他极化细胞，如动物神经元，假设微管和运动蛋白马达介导分泌小泡的长距离运输，而质膜上的短程运动由肌球蛋白和丝状肌动蛋白(F-肌动蛋白)组成的另一个系统介导。事实上，最近对许多丝状真菌的研究表明，微管、动蛋白、F-肌动蛋白和肌球蛋白对菌丝生长是必不可少的。然而，这些数据得出的大多数结论都是投机性的。这是因为分泌物的运动性从来没有被可视化，也没有对细胞中的整个马达进行系统的研究。在这个项目中，我们将应对这一挑战。我们已经开发了一种微观设置，使我们能够可视化包含甲壳素合成酶的单个囊泡的运动性。我们将标记许多其他分泌蛋白，包括分泌酶，并将监测它们向不断生长的菌丝顶端的输送。在使用红色和绿色荧光蛋白的共同定位研究中，我们将确定货物是在相同还是不同的运输小泡中旅行，从而阐明分泌的途径。我们将确定构成囊泡运动的细胞骨架元件，并利用现有的突变蛋白结构解决所有动蛋白和肌球蛋白马达在分泌中的作用。最后，我们将进一步探讨壳聚体双向运动的原因。这种行为是意想不到的，我们将调查这是否是分泌囊泡的一般特征。随后，我们将使用可光激活的荧光蛋白来表征这种运动性，以深入了解这一现象的原因。综上所述，我们将结合分子遗传学和生命细胞成像来：(1)确定菌丝细胞的分泌途径，(2)解决10个动蛋白和4个肌球蛋白在分泌小泡运动中的作用，(3)表征分泌小泡的双向运动，以了解这一行为背后的逻辑。该项目的预期结果将是对丝状真菌分泌途径的新见解。我们将全面了解货物到达菌丝顶端的途径，以及肌动蛋白和肌球蛋白在分泌囊泡传递中的作用。如果发现分泌囊泡的双向运动是一个普遍的特征，那么目前的分泌模式将不得不改变。因此，该项目将对真菌研究的各个方面产生根本的兴趣，但对于了解真菌的致病性和重组蛋白的工业化生产将特别重要。

Filamentous fungi are an evolutionarily successful group of organisms of enormous ecological importance as symbionts in mycorrhizal interactions with plants and decomposer of plant debris. They serve in industrial production of proteins and as pathogens pose a threat to public health and agriculture. The basic unit of a filamentous fungus is the hypha; this usually consists of a chain of elongated cells that grow by expansion at the tip, a process called tip growth. This mode of growth allows the invasion of tissue and substrate. Tip growth requires a continuous supply of newly synthesised membranes, proteins and cell wall precursors to the hyphal tip in a process named secretion. The carriers for these supplies are secretory transport vesicles that are taken to the tip where they fuse with the plasma membrane. It is important in the context of this grant application to note that our current view of secretion is that post-Golgi vesicles travel uni-directionally to the hyphal tip where they fuse with the plasma membrane, thereby delivering membranes and proteins. The mechanism by which secretory vesicles are delivered to the hyphal tip is not clear. In analogy to other polarised cells, such as animal neurons, it is assumed that microtubules and kinesin motors mediate long-distance transport of secretory vesicles, whereas short range motility at the plasma membrane is mediated by another system, consisting of myosins and filamentous actin (F-actin). Indeed, recent studies on numerous filamentous fungi, including the plant pathogen Ustilago maydis, demonstrated that microtubules, kinesins, F-actin and myosins are essential for hyphal growth. However, most conclusions from these data are speculative. This due to the fact that motility of secretory cargo was never visualised and no systematic studies on the whole repertoire of motors in a cell have been undertaken. In this project we will address this challenge. We have developed a microscopic setup that allows us to visualise the motility of individual chitin synthase-containing vesicles. We will label numerous other secretory proteins, including secreted enzymes, and will monitor their delivery to the growing hyphal tip. In co-localisation studies using red and green fluorescent proteins we will determine whether cargo travels in the same or different transport vesicles, thereby elucidating the pathways of secretion. We will determine the cytoskeletal elements that underlie vesicle motility and address the role of all kinesin and myosin motors in secretion by making use of existing mutant protein constructs. Finally, we will further investigate the reason for the bi-directional motility of chitosomes. This behaviour is unexpected and we will investigate whether it is a general feature of secretory vesicles. Subsequently, we will use photoactivatable fluorescent proteins to characterise this motility in order to get an insight into the reason for this phenomenon. In summary, we will combine molecular genetics and life cell imaging to: (1) determine the pathways of secretion in the hyphal cell, (2) address the role of 10 kinesins and 4 myosins in motility of secretory vesicles, (3) characterise the bi-directional motility of secretory vesicles in order to get to an understanding the logic behind this behaviour. The expected outcome of this project will be novel insights into the secretory pathway in filamentous fungi. We will provide a comprehensive understanding of the pathways by which cargo reaches the hyphal tip and of the role of kinesins and myosins in delivery of secretory vesicles. If it is discovered that bi-directional motility of secretory vesicles is a general feature, the current paradigm for secretion will have to be modified. This project will therefore be of fundamental interest to all aspects of fungal research, but it will be of particular importance in understanding fungal pathogenicity and industrial production of recombinant proteins.